Immunohistochemistry, or IHC, refers to the process of detecting, localizing, and quantifying antigens, such as a protein, in a biological sample, such as a tissue, and using specific binding moieties, such as antibodies specific to the particular antigens. This detection technique has the advantage of being able to show exactly where a given protein is located within the tissue sample. It is also an effective way to examine the tissues themselves. In situ hybridization, or ISH, refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on various biological samples, such as tissue (e.g., fresh frozen, formalin fixed paraffin embedded) and cytological samples. Upon recognition of the targets, whether the targets be nucleic acids or antigens, the recognition event can be detected through the use of various labels (e.g., chromogenic, fluorescent, luminescent, radiometric).
In situ hybridization (ISH) on tissue includes detecting a nucleic acid by applying a complementary strand of nucleic acid to which a reporter molecule is coupled. Visualization of the reporter molecule allows an observer to localize specific DNA or RNA sequences in a heterogeneous cell population, such as a histological, cytological, or environmental sample. Presently available ISH techniques include silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH).
Interrogation of gene expression in tissue sections using PCR or microarrays has been successfully used to classify patients' likelihood of tumor recurrence and identify those who may benefit from specific therapies. However, tissue specificity and cellular context, which improve the value of tissue-based assays, are lost during the mRNA extraction for PCR or microarray analysis. Moreover, false positive or negative results may be generated from the presence of “contaminating” non-tumor cells in the section. As such, there is a need for automated in situ hybridization assays which target mRNA (mRNA-ISH) that enables robust and reproducible evaluation of biomarker expression while preserving tissue context and specificity, as well as cell-cell relationships.
Chromogenic substrates have been used widely for immunohistochemistry for many years and for in situ hybridization more recently. Chromogenic detection offers a simple and cost-effective method of detection. Traditionally, chromogenic substrates precipitate when activated by the appropriate enzyme. That is, the traditional chromogenic substance is converted from a soluble reagent into an insoluble, colored precipitate upon contacting the enzyme. The resulting colored precipitate requires no special equipment for processing or visualizing. There are several qualities that successful IHC or ISH chromogenic substrates share. First, the substance should precipitate to a colored substance, preferably with a very high molar absorptivity. The enzyme substrate should have high solubility and reagent stability, but the precipitated chromogen products should be very insoluble, preferably in both aqueous and alcohol solutions. Enzyme turnover rates should be very high so as to highly amplify the signal from a single enzyme in a short amount of time. Particular limitations of current chromogenic techniques include the ability to multiplex, incompatibility towards post-staining processing (e.g., solvent washes, drying, subsequent staining), and limited color options.
For in situ assays, such as ISH assays and IHC assays, of tissue and cytological samples, especially multiplexed assays of such samples, it is highly desirable to identify and develop methods that provide desirable results without background interference. Tyramide Signal Amplification (TSA) is a known method based on catalyzed reporter deposition (CARD). U.S. Pat. No. 5,583,001 discloses a method for detection or quantitation of an analyte using an analyte-dependent enzyme activation system relying on catalyzed reporter deposition to amplify the reporter signal enhancing the catalysis of an enzyme in a CARD or TSA method by reacting a labeled phenol molecule with an enzyme. While tyramide signal amplification is known to amplify the visibility of targets, it is also associated with elevated background staining (e.g., amplification of non-specific recognition events).